The present invention relates to novel microorganisms having an ability to produce xylitol or D-xylulose, and a method for producing xylitol or D-xylulose by using a microorganism having an ability to produce xylitol or D-xylulose. D-Xylulose is useful as a material for the production of xylitol, and xylitol is useful as a sweetener in the field of food industry and the like.
The demand of xylitol which is a naturally occurring sugar alcohol is expected to increase in future. Xylitol is a promising low-calorie sweetener because it has lower calories and exhibits comparable sweetness compared with sucrose. In addition, because of its anti-dental caries property, it is utilized as a dental caries preventive sweetener. Furthermore, because xylitol does not elevate glucose level, it is utilized for fluid therapy in the treatment of diabetes. For these reasons, it is expected that the demand of xylitol will increase in future.
The current industrial production of xylitol mainly relies on hydrogenation of D-xylose as disclosed in U.S. Pat. No. 4,008,285. D-Xylose used as a raw material is obtained by hydrolysis of plant materials such as trees, straws, corn cobs, oat hulls and other xylan-rich materials.
However, such D-xylose produced by hydrolysis of plant materials suffers a drawback that it is rather expensive, and it is arisen from high production cost. For example, the low yield of the hydrolysis treatment of plant materials leads to low purity of the produced D-xylitol. Therefore, the acid used for the hydrolysis and the dyes must be removed by ion exchange treatment after the hydrolysis treatment, and the resulting D-xylose must be further crystallized to remove other hemicellulosic saccharides. In order to obtain D-xylose suitable for foodstuffs, further purification would be required. Such ion exchange treatment and crystallization treatment invite the increase of production cost.
Therefore, several methods for producing xylitol have been developed, which utilize readily available raw materials and generate little waste. For example, there have been developed methods for producing xylitol utilizing other pentitols as a starting material. One of such readily available pentitols is D-arabitol, and D-arabitol can be produced by using yeast (Can. J. Microbiol., 31, 1985, 467-471; J. Gen. Microbiol., 139, 1993, 1047-54). As a method for producing xylitol by utilizing D-arabitol as a raw material, there can be mentioned the method reported in Applied Microbiology., 18, 1969, 1031-1035, which comprises producing D-arabitol from glucose by fermentation using Debaryomyces hansenii ATCC20121, then converting the D-arabitol into D-xylulose using Acetobacter suboxydance, and converting D-xylulose into xylitol by the action of Candida guilliermondii var. soya. 
EP 403 392A and EP421 882A disclose methods comprising producing D-arabitol by fermentation using an osmosis-resistant yeast, then converting D-arabitol into D-xylulose using a bacterium belonging to the genus Acetobacter, the genus Gluconobacter, or the genus Klebsiella, forming a mixture of xylose and D-xylulose from the D-xylulose by the action of glucose (xylose) isomerase, and converting the obtained mixture of xylose and D-xylulose into xylitol by hydrogenation. There is also disclosed the production of xylitol comprising preliminarily concentrating xylose in the mixture of xylose and D-xylulose and converting the xylose into xylitol by hydrogenation.
However, those methods for the production of xylitol mentioned above utilize D-arabitol produced by fermentation as a starting material, and convert it by multiple process steps. Therefore, the processes are complicated, and less satisfactory ones in view of process economy compared with the methods based on extraction.
Accordingly, there has been desired a microorganism which has an ability to produce xylitol or D-xylulose through a single step by fermentation starting from glucose as used in the production of other saccharides and sugar alcohols. However, such a bacterium having an ability to produce xylitol or D-xylulose has not been reported so far.
On the other hand, breeding of xylitol fermenting bacteria has been attempted by using gene manipulation techniques. International Publication W094/10325 discloses production of xylitol from glucose by fermentation by using a recombinant microorganism obtained by introducing an arabitol dehydrogenase gene derived from a bacterium belonging to the genus Klebsiella and a xylitol dehydrogenase gene derived from a bacterium belonging to the genus Pichia into an arabitol fermenting microorganism (yeast belonging to the genus Candida, the gunus Torulopsis, or the genus Zygosaccharomyces). However, while production of 15 g/L of xylitol from 400 g/L of glucose has been reported for the aforementioned recombinant microorganism, it does not reach a practically useful accumulation level. Moreover, the aforementioned recombinant microorganism is introduced with a gene derived from a different species, and therefore information about its safety cannot be considered sufficient.
The present invention has been accomplished in view of the aforementioned state of the art, and its object is to provide a microorganism having an ability to produce xylitol or D-xylulose from glucose by fermentation, as well as a method for producing xylitol or D-xylulose utilizing such a microorganism.
In order to achieve the aforementioned object, the present inventors searched a microorganism having an ability to produce xylitol or D-xylulose from glucose by fermentation. As for direct production of sugar alcohols by fermentation of microorganisms such as yeasts, there have also been reported production of glycerol by using Zygosaccharomyces acidifaciens (Arch. Biochem., 7, 257-271 (1945)), production of erythritol by using a yeast belonging to the genus Trychosporonoides (Trychosporonoides sp., Biotechnology Letters, 15, 240-246 (1964)) and the like, in addition to the aforementioned arabitol fermentation. All of these yeasts having sugar alcohol producing ability show osmophilicity, i.e., good growth in a culture medium of high osmotic pressure. Therefore, while any microbes having xylitol producing ability have not found among the osmophilic yeasts, the present inventors considered that a novel microorganism having xylitol producing ability may exist among osmophilic microorganisms, and extensively screened osmophilic microorganisms. As a result, they found microorganisms having an ability to produce xylitol and D-xylulose from glucose among osmophilic microorganisms. Those microorganisms were estimated to be novel bacteria from the viewpoint of taxonomic phylogeny based on the nucleotide sequence of 16S rRNA gene. The present invention has been accomplished based on the aforementioned finding.
Accordingly, the present invention provides a microorganism belonging to the family Acetobacteracea, which has a 16S rRNA gene comprising a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence equivalent to the nucleotide sequence of SEQ ID NO: 1 from the viewpoint of molecular taxonomy based on the 16S rRNA sequence, and has an ability to produce xylitol or D-xylulose from glucose, and
A microorganism which has a 16S rRNA gene comprising a nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence of SEQ ID NO: 2 equivalent to the nucleotide sequence from the viewpoint of molecular taxonomy based on the 16S rRNA sequence, and has an ability to produce xylitol or D-xylulose from glucose.
Examples of the aforementioned microorganisms include, for example, those microorganisms belonging to the genus Asaia or the genus Zucharibacter, more specifically strains of Asaia ethanolifaciens or Zucharibacter floricola. Asaia ethanolifaciens is a new species (sp. nov.) provisionally designated by the present inventors. The genus Zucharibacter and Zucharibacter floricola are a new genus (gen. nov.) and new species, respectively, which were provisionally designated by the present inventors.
Particular examples of the aforememtioned microorganisms include, for example, strain P528 (FERM BP-6751), strain S877 (EERM BP-6752), strain S1009 (FERM BP-6753), strain S1019 (FERM BP-6754), and strain S1023 (FERM BP-6755).
The 16S rRNA gene of the strain P528 comprises the nucleotide sequence of SEQ ID NO: 1, and the 16S rRNA gene of the strain S877 comprises the nucleotide sequence of SEQ ID NO: 2. Partial sequences of the 16S rRNA gene of the strains S1009, S1019, and S1023 are of SEQ ID NOS: 3-5, respectively. These nucleotide sequences are equivalent to the nucleotide sequence of SEQ ID NO: 2 from the viewpoint of molecular taxonomy based on the nucleotide sequence of the 16S rRNA.
The present invention also provides a method for producing xylitol or D-xylulose, which comprises culturing a microorganism having an ability to produce xylitol or D-xylulose from glucose in a suitable medium to accumulate xylitol or D-xylulose in the medium, and collecting xylitol or D-xylulose from the medium.
Examples of the microorganism used for the above method includes, for example, a microorganism belonging to the family Acetobacteracea, which has a 16S rRNA gene comprising a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence equivalent to the nucleotide sequence from the viewpoint of molecular taxonomy based on the 16S rRNA sequence, and has an ability to produce xylitol or D-xylulose from glucose, and a microorganism belonging to the family Acetobacteracea, which has a 16S rRNA gene comprising a nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence equivalent to the nucleotide sequence from the viewpoint of molecular taxonomy based on the 16S rRNA sequence, and has an ability to produce xylitol or D-xylulose from glucose.
Specific examples of the aforementioned microorganisms include, for example, those microorganisms belonging to the genus Asaia or the genus Zucharibacter, more specifically strains of Asaia ethanolifaciens or Zucharibacter floricola. Particular examples of the aforementioned microorganisms include, for example, the strains P528, S877, S1009, S1019, and S1023.
The present invention further provides a method for producing ethanol, which comprises culturing the microbial strain P528 (FERM BP-6751) in a suitable medium to accumulate ethanol in the medium, and collecting ethanol from the medium.
According to the present invention, xylitol or D-xylulose can be efficiently produced from inexpensive materials such as glucose.
Further, ethanol can be produced by using the strain P528.